U.S. patent number 9,402,917 [Application Number 13/262,706] was granted by the patent office on 2016-08-02 for methods for the induction of broadly anti-hiv-1 neutralizing antibody responses employing liposome-mper peptide compositions.
This patent grant is currently assigned to DUKE UNIVERSITY. The grantee listed for this patent is S. Munir Alam, Barton F. Haynes, Moses D. Sekaran, Xiaoying Shen, Georgia Tomaras. Invention is credited to S. Munir Alam, Barton F. Haynes, Moses D. Sekaran, Xiaoying Shen, Georgia Tomaras.
United States Patent |
9,402,917 |
Alam , et al. |
August 2, 2016 |
**Please see images for:
( Certificate of Correction ) ** |
Methods for the induction of broadly anti-HIV-1 neutralizing
antibody responses employing liposome-MPER peptide compositions
Abstract
The present invention relates in general, to a formulation
suitable for use in inducing anti-HIV-1 antibodies, and, in
particular, to a formulation comprising Toll Like Receptor (TLR)
agonists with HIV-1 gp41 membrane proximal external region (MPER)
peptide-liposome conjugates for induction of broadly reactive
anti-HIV-1 antibodies. The invention also relates to methods of
inducing neutralizing anti-HIV-1 antibodies using such
formulations.
Inventors: |
Alam; S. Munir (Durham, NC),
Haynes; Barton F. (Durham, NC), Sekaran; Moses D.
(Durham, NC), Tomaras; Georgia (Durham, NC), Shen;
Xiaoying (Durham, NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
Alam; S. Munir
Haynes; Barton F.
Sekaran; Moses D.
Tomaras; Georgia
Shen; Xiaoying |
Durham
Durham
Durham
Durham
Durham |
NC
NC
NC
NC
NC |
US
US
US
US
US |
|
|
Assignee: |
DUKE UNIVERSITY (Durham,
NC)
|
Family
ID: |
42828908 |
Appl.
No.: |
13/262,706 |
Filed: |
April 5, 2010 |
PCT
Filed: |
April 05, 2010 |
PCT No.: |
PCT/US2010/001017 |
371(c)(1),(2),(4) Date: |
February 01, 2012 |
PCT
Pub. No.: |
WO2010/114628 |
PCT
Pub. Date: |
October 07, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120128758 A1 |
May 24, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61166625 |
Apr 3, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
39/12 (20130101); A61K 39/21 (20130101); A61P
31/12 (20180101); A61K 47/6911 (20170801); A61K
38/212 (20130101); A61P 37/04 (20180101); A61K
47/646 (20170801); A61P 31/18 (20180101); A61K
38/212 (20130101); A61K 2300/00 (20130101); A61K
2039/55555 (20130101); A61K 2039/55511 (20130101); C12N
2740/16134 (20130101); A61K 2039/55561 (20130101); A61K
2039/545 (20130101); A61K 9/1272 (20130101); A61K
2039/55572 (20130101) |
Current International
Class: |
A61K
39/385 (20060101); A61K 9/127 (20060101); A61P
31/18 (20060101); A61K 38/21 (20060101); A61K
47/48 (20060101); A61K 39/12 (20060101); A61K
39/21 (20060101); A61K 39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1250933 |
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Oct 2002 |
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EP |
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2006-512391 |
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Apr 2006 |
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JP |
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WO-95/25124 |
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Sep 1995 |
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WO |
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WO-2004/087738 |
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Oct 2004 |
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WO |
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WO-2006/110831 |
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Oct 2006 |
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WO |
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2008127651 |
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Oct 2008 |
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WO |
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WO-2009/111304 |
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Sep 2009 |
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WO |
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WO-2010/042942 |
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Apr 2010 |
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WO |
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WO-2010/045613 |
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Apr 2010 |
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WO |
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WO-2010/114628 |
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Oct 2010 |
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WO |
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WO-2010/114629 |
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Oct 2010 |
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WO |
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|
Primary Examiner: Parkin; Jeffrey
Attorney, Agent or Firm: Wilmer Cutler Pickering Hale and
Dorr LLP
Government Interests
This invention was made with government support under Grant No. AI
067854 awarded by the National Institutes of Health. The government
has certain rights in the invention.
Parent Case Text
This application is the U.S. national phase of International
Application No. PCT/US2010/001017, filed 5 Apr. 2010, which
designated the U.S. and claims the benefit of U.S. Provisional
Application No. 61/166,625, filed 3 Apr. 2009, the entire contents
of each of which are hereby incorporated by reference.
Claims
What is claimed is:
1. A method of inducing the production in a subject of anti-HIV-1
antibodies comprising administering to said subject a composition
comprising a liposome-peptide conjugate in an amount sufficient to
effect said induction, wherein said peptide comprises SEQ ID NO:17
(NEQELLELDKWASSWNWFNITNWLWYIK) presented on the surface of said
liposome.
2. The method according to claim 1 wherein said peptide further
comprises a hydrophobic linker.
3. The method according to claim 2 wherein said linker is
C-terminal to said MPER epitope.
4. The method according to claim 2 wherein said linker is GTH1.
5. The method according to claim 1 wherein said peptide is
NEQELLELDKWASSWNWFNITNWLWYIK (SEQ ID NO: 17) presented on the
surface of the liposome via the GTH1 linker.
6. The method according to claim 1 wherein the composition further
comprises an adjuvant.
7. The method according to claim 6 wherein said adjuvant is a Toll
Like Receptor (TLR) ligand.
8. The method according to claim 7 wherein said TLR ligand is a
TLR9 ligand.
9. The method according to claim 8 wherein said TLR9 ligand is
oligo CpG.
10. The method according to claim 7 wherein said TLR ligand is a
TLR7/8 ligand.
11. The method according to claim 10 wherein said TLR7/8 ligand is
R-848.
12. The method according to claim 7 wherein said TLR ligand is a
TLR4 ligand.
13. The method according to claim 12 wherein said TLR4 ligand is
monophosphorylipid A.
14. The method according to claim 7 wherein said conjugate
comprises a TLR9 ligand and a TLR7/8 ligand.
15. The method according to claim 14 wherein said TLR9 ligand is
oligo CpG and said TLR7/8 ligand is R-848.
16. The method according to claim 7 wherein said conjugate
comprises a TLR9 ligand and a TLR4 ligand.
17. The method according to claim 16 wherein said TLR9 ligand is
oligo CpG and said TLR4 ligand is R-848.
18. The method according to claim 7 wherein said composition
further comprises interferon-.alpha.encapsulated therewithin.
19. The method according to claim 7 wherein said composition is
administered as a prime or a boost.
20. A composition comprising a liposome and an MPER peptide
comprising the peptide of SEQ ID NO:17, wherein the peptide is
presented on the surface of the liposome via a hydrophobic
linker.
21. The composition according to claim 20 further comprising
interferon-.alpha. encapsulated within said liposome.
22. The composition of claim 20 further comprising an adjuvant.
23. The composition of claim 21, wherein the adjuvant is a Toll
Like Receptor (TLR) ligand.
24. The composition of claim 21, wherein the TLR ligand is TLR4,
TLR7/8, TLR9, or any combination thereof.
25. The composition of claim 24, wherein the TLR4 ligand is
monophosphorylipid A.
26. The composition of claim 24, wherein the TLR7/8 ligand is
R848.
27. The composition of claim 24, wherein the TLR9 ligand is
oligoCpG.
28. The composition of claim 24, wherein the linker is GTH1.
Description
TECHNICAL FIELD
The present invention relates in general, to a formulation suitable
for use in inducing anti-HIV-1 antibodies, and, in particular, to a
formulation comprising Toll Like Receptor (TLR) agonists with HIV-1
gp41 membrane proximal external region (MPER) peptide-liposome
conjugates for induction of broadly reactive anti-HIV-1 antibodies.
The invention also relates to methods of inducing neutralizing
anti-HIV-1 antibodies using such formulations.
BACKGROUND
One of the major challenges to HIV-1 vaccine development has been
the inability of immunogens to induce broadly neutralizing
antibodies (nAb). nAbs are generated during HIV-1 infection.
However, most of the nAbs generated neutralize only the autologous
viruses or closely related strains (Moog et al, J. Virol.
71:3734-3741 (1997), Gray et al, J. Virol. 81:6187-6196 (2007)).
HIV envelope (Env) constantly mutates to escape from existing nAb
response (Albert et al, Aids 4:107-112 (1990), Wei et al, Nature
422:307-312) (2003)). nAb responses do evolve over the course of
the HIV infection. However, with the mutation capacity of HIV-1
viruses, neutralizing antibody responses always seem to "lag
behind" virus evolution (Wei et al, Nature 422:307-312 (2003)),
Richman et al, Proc. Natl. Acad. Sci. USA 100:4144-4149 (2003),
Geffin et al, Virology 310:207-215 (2003)).
After extensive research, a handful of broadly neutralizing
monoclonal antibodies (mAbs) against HIV have been identified
(Buchacher et al, AIDS Res. Hum. Retroviruses 10:359-369 (1994),
Zwick et al, J. Virol. 75:10892-10895 (2001), Burton et al, Proc.
Natl. Acad. Sci. USA 888:10134-10137 (1991)). Two such antibodies,
2F5 and 4E10, target the conserved membrane-proximal external
region (MPER) of HIV, have a broad spectrum of neutralization
(Binley et al, J. Virol. 78:13232-13252 (2004)), and have been
shown to neutralize 80% and 100% of newly transmitted viruses
(Mehandru et al, J. Virol. 78:14039-14042 (2004)), respectively.
When passively administered in combination with several other
broadly neutralizing monoclonal antibodies, a cocktail of mAbs
composed of 2G12, 2F5 and 4E10 successfully protected the host from
virus infection in animal models (Baba et al, Nat. Med. 6:200-206
(2000), Ferrantelli et al, J. Infect. Dis. 189:2167-2173 (2004),
Mascola et al, Nat. Med. 6:207-210 (2000), Ruprecht et al, Vaccine
21:3370-3373 (2003)), or delayed virus rebound after cessation of
antiretroviral therapy (Trkola et al, Nat. Med. 11:615-622
(2005)).
The potential of using 2F5 and 4E10 to prevent HIV infection is
greatly compromised by the fact that HIV infected patients rarely
develop these antibodies spontaneously (Dhillon et al, J. Virol.
81:6548-6562 (2007)), and there has been no success in inducing
2F5- and 4E10-like antibodies by vaccination (Kim et al, Vaccine
25:5102-5114 (2006), Coeffier et al, Vaccine 19:684-693 (2000),
Joyce et al, J. Biol. Chem. 277:45811-45820 (2002), Ho et al,
Vaccine 23:1559-1573 (2005), Zhang et al, Immunobiology 210:639-645
(2005)). Identification of subjects that develop 2F5- or 4E10-like
antibodies during natural HIV-1 infection, and developing an
understanding of the mechanism of, or hindrance to, these broadly
neutralizing antibodies is important for AIDS vaccine design.
The present invention results, at least in part, from the
identification and characterization of a rare Env mutation in the
HIV-1 MPER region which is associated with an increase in
neutralization sensitivity to 2F5 and 4E10 mAbs. The invention also
results from the development of constructs that can modulate B cell
tolerance and enhance antibody responses against poorly immunogenic
HIV-1gp41MPER epitopes.
SUMMARY OF THE INVENTION
In general, the present invention relates to a formulation suitable
for use in inducing anti-HIV-1 antibodies. More specifically, the
invention relates to a formulation comprising TLR agonists with
HIV-1 gp41MPER peptide-liposome conjugates, and to methods of
inducing broadly reactive neutralizing anti-HIV-1 antibodies using
same.
Objects and advantages of the present invention will be clear from
the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Neutralizing sensitivity of TND_669S and TND_669L
Env-pseudoviruses by autologous and heterologous sera/Ab.
SC42-15mo, SC42-27mo, and SC42-5yr are autologous sera from 15mo,
27mo, and 65 mo p.i.; SCO3-TT29 are heterologous sera from Trinidad
cohort; IBBJT, BD are HIV+ patient sera used as positive controls;
HIVIG is purified pooled IgG from HIV+ patient sera. Due to sample
availability limitation, not all samples were tested more than
once. For those samples that were tested more than once, the bars
represents the average titer, and the error bars represent standard
errors.
FIG. 2. Partial alignment of selected SC42 Env sequences. TND_669S,
TND_669L and 7534-xx (wherein "xx" is as shown in FIG. 2) are
sequences from 15 mo p.i. plasma; Other sequence shown are selected
sequences from week 0 (2661-x), week 1 (00SC42-xx) and from 60 mo
(95SC42-xx) (wherein "x" and "xx" are as shown in FIG. 2) p.i.
plasma. Consensus epitope sequences for 2F5 and 4E10 are
highlighted in blue and green boxes, respectively. FIG. 2 discloses
SEQ ID NOS 18-20, 20-22, 22-23, 21, 21-22, 24-25, 25 and 25,
respectively, in order of appearance.
FIGS. 3A and 3B. Neutralization of TND_669S and TND_669L
Env-pseudoviruses by various monoclonal antibodies and the entry
inhibitor T20. The mean IC50 of each reagent against the two
strains are shown in FIG. 3A, with error bars showing the standard
errors. The IC50 values and the fold differences of each
neutralizing antibodies in its potency against TND_669S and
TND_669L are shown in FIG. 3B. Each IC50 was obtained from at least
two independent tests. Data for 2F5, 4E10, TriMab, 1b12, and 2G12
also include one set of data from a test performed by Dr.
Montefiori's laboratory (Duke University). The fold difference
between the IC50 of each mAb against TND_669S and TND_669L
(TND_669:tND_669L) are listed in the last column of the table, and
the ones with significant increase in sensitivity of TND_669S are
highlighted in yellow (and marked with a " ").
FIGS. 4A and 4B. Peptide absorption neutralization assays.
Neutralization of the TND_669S Env-pseudovirus by mAb 2F5 was
tested with different doses of 2F5 peptides. Inhibition of 2F5 mAb
neutralization by the mutant peptide (containing 2F5 epitope with
the L669S mutation, 2F5.sub.656-670/L669S) is shown FIG. 4A. The
inhibition curves generated by the peptide containing the consensus
peptide (consensus peptide) are similar. The IC50 data are
summarized in the table in FIG. 4 B. Similar tests were also
performed on the TND_669L viruses. A similar trend was observed,
however, due to the low sensitivity of TND_669L to 2F5 mAb, data
generated using the TND_669L pseudovirus were not quantitative.
FIGS. 5A and 5B. BIAcore SPR assay for binding avidity of F5mut
(FIG. 5A) and F5con (FIG. 5B) peptides to mAb 2F5.
FIGS. 6A and 6B. Binding of 2F5 mAb to peptide-liposome conjugates.
FIG. 6A. Comparison of normalized specific binding responses of 2F5
mAb to 2F5 peptide-liposomes (broken line) and L669S mutant
peptide-liposomes (solid line). The inset shows the magnified image
of the dissociation phase of the 2F5 mAb interaction (120-400 s).
FIG. 6B. The encounter-docking model of 2F5 mAb-peptide-liposome
interactions and the estimated rate constants of association and
dissociation steps.
FIG. 7. Dual infection fitness assay in PBMC. Shown is a test with
input ratio of 9:1 (TND_669S:TND_669L). The relative fitness value
1+S=1.86. (1+S=exp(d)=exp{ ln
[(TM(t2).times.TL(t1))/(TL(t2).times.TM(t1))]/.DELTA.t}. Tests of 3
individual tests with different virus input ratios all conferred a
1+S value of 1.80.about.1.90.
FIG. 8. HIV-1 gp41 MPER peptides that include the epitopes of the
two broadly neutralizing antibodies 2F5 and 4E10. Amino acid
sequences of the gp41 MPER peptides (SEQ ID NOS 26, 9-10 and 16-17,
respectively, in order of appearance) that can be conjugated to
synthetic liposomes are shown.
FIG. 9. Structures of TLR agonists formulated with liposomes. A
schematic picture of the immunogen designs shows the
peptide-liposomes containing different TLR agonists as adjuvants;
TLR4 (Lipid A); TLR9 (oCpG) (SEQ ID NO: 27) and TLR7 (R848).
FIGS. 10A-10C. Interaction of 2F5 mAb with MPER peptide-liposomes
conjugated to TLR adjuvants. FIG. 10A shows strong binding of 2F5
mab to gp41 MPER liposome constructs with Lipid A (200 .mu.g dose
equivalent). FIG. 10B shows binding of 2F5 mAb to oCpG (50 .mu.g
dose equivalent) conjugated gp41 MPER liposomes. FIG. 10C shows
binding of 2F5 mAb to R848-conjugated gp41 MPER containing
liposomes. In comparison to control liposomes with only TLR
adjuvants, strong binding of 2F5 mAb was observed to each of the
gp41MPER-adjuvant liposomal constructs.
FIG. 11. IFN.alpha. encapsulated MPER peptide liposomes
FIG. 12. IFN.alpha. encapsulated liposome with multiple TLR
ligands. These constructs have the potential to provide synergy in
B cell responses via dual TLR triggering.
FIG. 13. Crystal structures of 2F5 (Ofek et al, J. Virol. 78:10724
(2004)) and 4E10 (Cardoso et al, Immunity 22:163-173 (2005)) and
design of mutations in the CDR H3 loop to eliminate binding to
lipids and HIV-1 viral membrane.
FIGS. 14A and 14B. Substitution of hydrophobic residues of 4E10
(FIG. 14A) and 2F5 (FIG. 14B) CDR H3 disrupt lipid binding and
abrogate ability of both mAbs to neutralize HIV-1.
FIG. 15. Neutralization of QZ4734 and QZ4734/L669S pseudotyped
viruses by 2F5 mAb (tested on TZM-b1 cells). QZ4734/L669S was
generated by introducing L669S single mutation into the QZ4734
envelope. Numbers by the curves indicate the IC50 values.
FIG. 16. Neutralization of TND_669S and two other stains (7534.2
and 7534.11) isolated from the same plasma sample (15 mo post
infection) by 2F5 and TriMab (1:1:1 combination of 2F5, 4E10 and
2G12). Numbers above each bar represents IC50 values. The test was
performed on TZM-b1 cells.
FIG. 17. Induction of gp41 MPER specific antibody (SEQ ID NO: 9)
responses in guinea pigs immunized with MPER liposomal
immunogens.
FIG. 18. Induction of gp41MPER specific antibody responses in Non
human primates (NHP) immunized with MPER liposomal immunogens.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a liposome-based adjuvant
conjugate that presents TLR ligands and HIV-1 gp41 neutralizing
antigens, and to a method of inducing neutralizing anti-HIV-1
antibodies in a subject (e.g., a human subject) using same.
Suitable neutralizing antigens include gp41MPER epitope peptides
(Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004),
Stiegler and Katinger, J. Antimicrob. Chemother. 512:757-759
(2003), Zwick et al, Journal of Virology 79:1252-1261 (2005),
Purtscher et al, AIDS10:587 (1996)) and variants thereof, for
example, variants that confer higher neutralization sensitivity to
MPER Mabs 2F5 and 4E10. In a preferred embodiment, the variant is a
MPER epitope peptide with an L669S mutation that confers higher
neutralization sensitivity to MPER mAbs 2F5 and 4E10 (Shen et al,
J. Virology 83: 3617-25 (2009)).
Liposomes suitable for use in the invention include, but are not
limited to, those comprising POPC, POPE, DMPA (or sphingomyelin
(SM)), lysophosphorylcholine, phosphatidylserine, and cholesterol
(Ch). While optimum ratios can be determined by one skilled in the
art, examples include POPC:POPE (or POPS):SM:Ch or POPC:POPE (or
POPS):DMPA:Ch at ratios of 45:25:20:10. Alternative formulations of
liposomes that can be used include DMPC
(1,2-dimyristoyl-sn-glycero-3-phosphocholine) (or
lysophosphorylcholine), cholesterol (Ch) and DMPG
(1,2-dimyristoyl-sn-glycero-3-phoshpho-rac-(1-glycerol) formulated
at a molar ratio of 9:7.5:1 (Wassef et al, ImmunoMethods 4:217-222
(1994); Alving et al, G. Gregoriadis (ed.), Liposome technology
2.sup.nd ed., vol. III CRC Press, Inc., Boca Raton, Fla. (1993);
Richards et al, Infect. Immun. 66(6):285902865 (1998)). The
above-described lipid compositions can be complexed with lipid A
and used as an immunogen to induce antibody responses against
phospholipids (Schuster et al, J. Immunol. 122:900-905 (1979)). A
preferred formulation comprises POPC:POPS:Ch at ratios of 60:30:10
complexed with lipid A according to Schuster et al, J. Immunol.
122:900-905 (1979).
In accordance with the invention, immune response enhancing TLR
ligands, for example, monophosphorylipid A (MPL-A, TLR4 ligand),
oligo CpG (TLR9 ligand) and R-848 (TLR7/8 ligand), are formulated
either individually or in combination into liposomes conjugated
with an HIV-1 gp41MPER peptide immunogen. A preferred combination
of TLR agonists comprises oCpG (TLR9) (Hemni et al, Nature
408:740-745 (2004)) and R848 (TLR7/8) (Hemni et al, Nat. Immunol.
3:196-200 (2002)).
Additional designs of constructs of the invention include MPER
peptide-liposome encapsulated with the cytokine interferon
(IFN)-.alpha. and either encapsulated or membrane bound CD40
ligand. Two broadly neutralizing gp41 MPER antibodies (2F5, 4E10)
bind with high affinity to such TLR ligand adjuvant-associated
liposome constructs. These constructs can be used to modulate B
cell tolerance, direct liposomes to certain B cell populations
capable of making broadly reactive neutralizing antibodies, and in
enhance antibody responses against poorly immunogenic HIV-1
gp41MPER epitopes.
Autoreactive B cells can be activated by TLR ligands through a
mechanism dependent on dual engagement of the B cell receptor (BCR)
and TLR (Leadbetter et al, Nature 416:603 (2002); Marshak-Rothstein
et al, Annu. Rev. Immunol. 25: 419-41 (2007), Herlands et al,
Immunity 29:249-260 (2008), Schlomchik, Immunity 28:18-28 (2008)).
In a preferred immunogen design of the instant invention, soluble
IFN-.alpha. is encapsulated into liposomes conjugated to MPER
peptides such as MPER656 or MPER656-L669S peptides. IFN-.alpha. has
been reported to modulate and relax the selectivity for
autoreactive B cells by lowering the BCR activation threshold
(Uccellini et al, J. Immunol. 181:5875-5884 (2008)). The design of
the immunogens results from the observation that lipid reactivity
of gp41MPER antibodies is required for both binding to membrane
bound MPER epitopes and in the neutralization of HIV-1.
The B cell subsets that the liposomes can target include any B cell
subset capable of making polyreactive antibodies that react with
both lipids and the gp41 epitopes of the MPER. These B cell subsets
include, but are not limited to, the marginal zone IgM+ CD27+ B
cell subset (Weill et al, Annu. Rev. Immunol. 27:267-85 (2009), Li
et al, J. Exp. Med 195: 181-188 (2002)), the transitional
populations of human B cells (Sims et al, Blood 105:4390-4398
(2005)), and the human equivalent of the B cells that express the
human equivalent of the mouse Immunoglobulin (Ig) light chain
lambda X (Li et al, Proc. Natl. Acad. Sci. 103:11264-11269 (2006),
Witsch et al, J. Exp. Med. 203:1761-1772 (2006)). All of these B
cell subsets have the capacity to make multireactive antibodies
and, therefore, to make antibodies that have the characteristic of
reacting with both lipids and HIV-1 gp41. That the liposomes have
the characteristic of having both lipids and gp41 in them, should
result in the selective targeting of these immunogens to the B
cells of interest. Because these liposomes can be used to
transiently break tolerance of B cells or to target rare B cell
subsets, it can be seen that other HIV-1 envelope immunogens, such
as deglycosylated envelope preparations, such as described below,
can be formulated in the liposomes containing TLR4 agonists, TLR7/8
agonists and IFN .alpha..
The deglycosylated JRFL gp 140 Env protein and the CD4-binding site
mutant gp140 (JRFL APA) have been described in a previous
application (see, for example, WO 2008/033500). Deglycosylated env
and Env mutated to not bind CD4 so as not to be immunosuppressive
can be anchored in the liposomes by incorporating a transmembrane
domain and, after solubilizing in detergent, can be reconstituted
into synthetic lipsomes. Alternatively, His-tagged (c-terminus end)
versions of the Env gp140 can be anchored into liposomes as
described for an intermediate form of HIV-1 gp41 (gp41-inter)
Given that many B cell subsets capable of making polyreactive
antibodies also bind mammalian DNA, addition of DNA to liposomes
can be used to target the immunogens to the responsive B cells.
The liposome-containing formulations of the invention can be
administered, for example, by intramuscular, intravenous,
intraperitoneal or subcutaneous injection. Additionally, the
formulations can be administered via the intranasal route, or
intrarectally or vaginally as a suppository-like vehicle.
Generally, the liposomes are suspended in an aqueous liquid such as
normal saline or phosphate buffered saline pH 7.0. Optimum dosing
regimens can be readily determined by one skilled in the art.
Certain aspects of the invention can be described in greater detail
in the non-limiting Examples that follows. See also Published PCT
Application Nos. WO 2006/110831 and WO 2008/127651, U.S. Published
Application Nos. 2008/0031890 and 2008/0057075, U.S. Provisional
Application No. 60/960,413 and U.S. application Ser. No.
11/918,219. (See also related applications entitled "Formulation",
filed Apr. 3, 2009 and "Mouse Model", filed Apr. 3, 2009 .
EXAMPLE 1
Experimental Details
Subjects
Trinidad Seroconverter Cohort was described preciously (Blattner et
al, J. Infect. Dis. 189:1793-1801 (2004)). Briefly, patients from a
sexually transmitted disease (STD) clinic were monitored for HIV
infection and enrolled upon seroconvertion. Infections occurred
through heterosexual contact and subtype B viruses accounted for
all the infections. The patient of interest in this study, SC42,
was naive for antiviral therapy until 5 yr into infection.
Molecular Cloning of Full-Length Envelopes
Cloning strategy of full-length gp160 has been described previously
(Wei et al, Nature 422:307-312 (2003), Li et al, J. Virol.
79:10108-10125 (2005)). Briefly, viral RNA was extracted from
patient plasma samples using QIAmp Viral RNA Mini Kit (Qiagen,
Valencia, CA) and subsequently reverse-transcribed into cDNA using
SuperScript II ((Invitrogen Corp., Carlsbad, CA) and random hexamer
primers. Full length envelope sequences were generated by nested
PCR with the following primers: 1st round primers 5'OUT
5'-TAGAGCCCTGGAAGCATCCAGGAAG- 3'(SEQ ID NO: 1), nt 5852-5876 and
3'OUT 5'-TTGCTACTTGTGATTGCTCCATGT-3'(SEQ ID NO: 2), nt 8912-8935);
and 2nd round primers 5'Intopo 5'-CACCTAGGCATCTCCTATGGCAGGAAGA
AG-3'(SEQ ID NO: 3), nt 5957-5982and 3'IN
5'-GTCTCGAGATACTGCTCCCACCC-3'(SEQ ID NO: 4), nt 8881-8903). The PCR
products were purified and then directly ligated into the
directional cloning vector pcDNA 3.1 D/V5-His-TOPO (Invitrogen)
following the manufacturer's directions. This pcDNA 3.1 D/V5-
His-TOPO vector contains a cytomegalovirus promoter that allows the
expression of envelope proteins for subsequent pseudovirus
production.
Mutagenesis for Introduction of Single Mutation
A QuikChange XL Site-directed Mutagenesis Kit (Invitrogen Corp) was
used to introduce S669L mutation into HS-MPER to generate
HS-MPERIS669L, and K665N mutation into HS-MPER to generate
HS-MPER/K665N following the manufactures instructions. The primers
for introducing S669L mutation into HS-MPER were: fN-MPER_S669L
(5'-ggataagtgggcaagtttgtggaattggtttgac-3'(SEQ ID NO: 5)) and
r7534.5_S669L (5'-GTCAAACCAATTCCACAAACTTGCCCACTTATCC3'(SEQ ID NO:
6)); the primers for introducing K665N into HS-MPER were:
fHS-MPER_K665N (5'-gaattattagaattggataaCtgggcaagttcgtgg3'(SEQ ID
NO: 7)) and r7534.5_K665N
(5'-CCACGAACTTGCCCAGTTATCCAATTCTAATAATTC3'(SEQ ID NO: 8)).
Production and Titration of Env-Pseudoviruses
Production and titration of the env-pseudoviruses was conducted
following procedures modified from methods previously described (Li
et al, J. Virol. 79:10108-10125 (2005)) with minor modifications.
Full-length env clones in pcDNA3.1D/V5-His-TOPO vector were
co-transfected into 293T cells with an env-deficient HIV-1 backbone
(pSG3.DELTA.env) using FuGENE.RTM. HD transfection reagent (Roche
Applied Science, Basel, Switzerland). Tissue culture fluid was
harvested after 24-36 h of incubation and fresh fetal bovine serum
was added to the virus stock to make a final concentration of
20%.
The 50% tissue culture infectious dose (TCID50) of each virus
preparation was determined on JC53-BL cells as previously described
(Li et al, J. Virol. 79:10108-10125 (2005)). Briefly, serial
diluted virus stocks were used to infect JC53-BL cells on
96-well-flat-bottom-plates for 48 h. The cells were then lysed with
and the relative luminescence units (RLU) determined by
BriteLite.TM. assay system (PerkinElmer, Inc., Waltham, Mass.).
Wells with luciferase luminescence 2.5-fold over that of the cells
only control were considered positive for virus infection. TCID50
was calculated using the Reed-Muench formula.
Neutralization Assay
Neutralization assays for the pseudoviruses were performed on
JC53-BL cells on 96-well-flat-bottom-plates as previously described
(Li et al, J. Virol. 79:10108-10125 (2005)). Briefly, serially
diluted serum samples or purified Abs were incubated with testing
viruses, followed by addition of JC53-BL cells. The relative
luminescence unit (RLU) of each well was measured with
BriteLite.TM. assay system and the IC50 was determined as the
highest dilution of serum (in cases of serum samples) or the lowest
concentration of Ab (in cases of purified Abs) that was able to
inhibit virus infection by 50% compared to the virus control.
Peptide Absorption Neutralization Assay
Peptide absorption neutralization assay was modified from
neutralization assay. Serially diluted serum samples or purified
Abs were pre-incubated with properly diluted peptide for 1 h before
addition of virus, followed by regular neutralization assays.
Surface Plasmon Resonance (SPR) Assays
SPR binding assays were performed on a BlAcore 3000 (BlAcore Inc,
Piscattaway, NJ) maintained at 20.degree. C. as previously
described (Alam et al, J. Immunol. 178:4424-4435 (2007)).
Biotinylated versions of SP62 peptides- gp41 652-671
(QQEKNEQELLELDKWASLWN (SEQ ID NO: 9)) and SP62-L669S (gp41 652-671)
(QQEKNEQELLELDKWASSWN (SEQ ID NO: 10)), and control peptides with
scrambled sequences (2F5.sub.656-670 Scrambled and
2F5.sub.656-670/L669SScrambled), were individually anchored on a
BlAcore SA sensor chip as described (Alam et al, J. Immunol.
178:4424-4435 (2007), Alam et al, AIDS Res. Hum. Retroviruses
20:836-845(2004)). Each peptide was injected until 100 to 150
response unit (RU) of binding to streptavidin was observed.
Specific binding responses of mAb binding were obtained following
subtraction of non-specific binding on the scrambled 2F5 peptide
surface. Rate constants were measured using the bivalent analyte
model (to account for the avidity of bivalent Ig molecules) and
global curve fitting to binding curves obtained from 2F5
titrations, which ranged from 0.01to 119 nM for mAb 2F5.mAb 2F5
were injected at 30 uL/min for 2-6 min and Glycine-HCI pH 2.0and
surfactant P20 (0.01%) were used as the regeneration buffer.
SPR assay with liposome-anchored peptides were done in a similar
fashion as described above. The peptides used are SP62 (gp41
652-671)-GTH1 (QQEKNEQELLELDKWASLWNYKRWIILGLNKIVRMYS-biotin,
containing the consensus 2F5 epitope (SEQ ID NO: 11)) and
SP62-L669S (gp41 652-671)-GTH1
(QQEKNEQELLELDKWASSWNYKRWIILGLNKIVRMYS-biotin, containing the 2F5
epitope with the L669S substitution (SEQ ID NO: 12)).
Fitness Assay
The dual infection fitness assay was performed as previously
described (Lu et al, J. Virol. 78:4628-4637 (2004)) with minor
modifications. HIV-1 infectious chimeric viruses containing
TND_669S or TND_669L env and a marker sequence (either Salmonella
enterica serovar Typhimurium histidinol dehydrogenase [hisD] gene
or the human placental heat-stable alkaline phosphatase [PLAP]
gene) were generated by cotranfecting env PCR product and NL4-3
background vector with a reporter gene. In a dual infection fitness
assay, two chimeric viruses with specific input ratio (as
determined by real-time PCR of the reporter genes) were used to
co-infect PBMC (MOI=0.001). Relative production of the viruses with
the two Env species in the culture were measured by the
corresponding marker (hisD or PLAP) using real-time RT-PCR.
Production of an individual virus in a dual infection was
determined by calculating the percentage of the individual virus in
the total virus population at specific time points (Day 4, 7, and
10). The relative fitness value (1+S) of the individual virus was
determined by following equation as previously described (Wu et al,
J. Virol. 80:2380-2389 (2006)): (1+S=exp(d)=exp{ln[(TM
(t2).times.TL(t1))/(TL (t2).times.TM(t1))]/.DELTA.t}
1+S=exp, where S is the selection coefficient; M.sub.t, M.sub.0,
L.sub.t, and L.sub.0 are the proportion of more fit variant or less
fit variant at time point t and the initial proportion (0) in the
inoculum respectively.
Results
Identification of TND_669S Envelope
Multiple longitudinal Env clones were obtained from plasma samples
of SC42, NL4-3 Env-pseudotyped viruses were made from the Env
clones, and neutralizing sensitivity of selected Env clones against
autologous as well as heterologous sera was tested. An envelope
strain that was highly sensitive to neutralization by autologous
sera was identified. TND_669S, an envelope clone obtained from a
chronically infected HIV+ subject showed unexpectedly high
sensitivity to neutralization by both autologous and heterologous
sera. TND_669S was neutralized by contemporaneous and 27 month
(post enrollment) autologous sera with titers of 845 and 1,353
respectively, while TND_669L, another isolate the neutralization
sensitivity of which was typical of envelope clones obtained from
the same time point (15 month post enrollment) and was
retrospectively selected for comparison based on its envelope
sequence, was not sensitive to contemporaneous autologous serum
neutralization and was neutralized by 27 months post enrollment
autologous serum with a titer of only 26 (FIG. 1). TND_669S and
TND_669L Env-pseudoviruses were then tested against a panel
heterologous patient sera as well as several HIV+ sera/Ab used as
positive controls. TND_669S Env-pseudovirus was shown to be up to
47-fold more sensitive to neutralization by heterologous sera
within Trinidad cohort. Among the 14 patient sera tested, 7
neutralized the TND_669S pseudovirus more than 10-fold more
efficiently than the TND_669L pseudovirus (FIG. 1).
Identification of the L669S Mutation
The protein and DNA sequences for TND_669S and TND_669L gp 160 were
examined for genetic variations responsible for the increased
neutralizing sensitivity of TND_669S envelope. There are 6
nucleotide differences between the two env DNA sequences. However,
5 of those are synomonous mutations, resulting in a single amino
acid difference between TND_669S and TND_669L Env. The single amino
acid difference is located at position 669, near the C-terminus of
the 2F5 epitope and 2 aa upstream of the 4E10 epitope in the MPER
(FIG. 2). TND_669L contains the 2F5 consensus sequence while
TND_669S contains a L669S mutation. 3 out of 10 clones obtained
from the 15 month post enrollment plasma of patient SC42 contain
this mutation, while this mutation was not found in either 1 wk
post enrollment plasma or 5 yr post enrollment plasma.
Interestingly, only 1 out of around 1000 full-length Env sequences
in LANL database contains this L669S mutation.
Sensitivity of the L669s Mutant to Monoclonal Antibodies
Based on the location of the L669S mutation, sensitivity of the
TND_669S and TND_669L to 2F5 and 4E10 mAbs was tested. Not
surprisingly, TND_669S was highly sensitive to 2F5 mAb while
TND_669L was only moderately sensitive (FIG. 3). Interestingly,
TND_669S is also highly sensitive to neutralization by 4E10 mAb
compared to TND_669L. As shown in FIG. 3, the IC.sub.50 of 2F5 and
4E10 mAbs against TND_669S Env-pseudovirus were 279- and 275-fold
lower than that against TND_669L Env-pseudovirus, respectively. The
mean IC.sub.50 of TND_669S and TND_669L were 0.014 (.+-.0.0056) and
3.92 (.+-..sup..about.1.52) respectivelym for 2F5, and 0.031
(.+-.0.012) and 8.49 (.+-.1.29) .mu.g/ml, respectively, for
4E10.
Sensitivity of TND_669S and TND_669L pseudoviruses to several other
neutralizing agents, including the glycan dependent mAb 52D and the
entry inhibitor T20 was also tested (FIG. 3). No significant
difference in sensitivity to 2G12 and T20 and only a slight
increase in sensitivity to 17b and 1b12 (-2 and 4-fold,
respectively) was observed for the TND_669S pseudovirus, indicating
that global changes in envelope, if any, can not account for the
dramatically enhanced neutralizing sensitivity observed for the
TND_669S envelope. Differences in sensitivity of the two strains
against 1.7B, 23E, and E51 could not be quantified because the
TND_669L is not sensitive enough to neutralization by these
antibodies. Interestingly, the TND_669L envelope was also not
sensitive to 447-52D neutralization while the TND_669L envelope was
neutralized with an IC.sub.50 of 0.31 .mu.g/ml, indicating an
enhancement of >161-fold in 447-52D sensitivity associated with
the L669S mutation.
Single L669S Mutation Accounts for the Phenotypic Change
To confirm that the L669S mutation alone is responsible for the
phenotypic change, a S669L mutation was introduced into the
TND_669S envelope by site-directed mutagenesis. The resulting
TND_669S/S669L showed only moderate sensitivity to 2F5 comparable
to that of TND_669L, confirming the sole contribution of the L669S
mutation in the TND 669S to the increased sensitivity to
neutralization. Next, the role of the virus backbone in the
phenotypic change associated with the L669S mutation was
investigated. A L669S mutation was introduced into the envelope of
another primary isolate, QZ4734. The L669S mutation rendered the
QZ4734 Env-pseudovirus more than two logarithmic magnitudes more
sensitive to neutralization by the 2F5 mAb (FIG. 15). Furthermore,
the other two clones that share the L669S mutation showed similar
magnitude of increase in sensitivity against 2F5 (FIG. 16). These
findings suggest that the L669S can increase the sensitivity of
HIV-1 envelope to neutralization by MPER antibodies regardless of
the virus background.
Neutralizing of TND_669S Envelope is Mediated by 2F5 Binding to its
Conventional Epitope
Characterization of a 2F5-resistent Env variant has shown that a
K665N mutation in the DKW core region abrogates 2F5 binding and
results in 2F5 resistance (Purtscher et al, Aids 10:587-593
(1996)). This suggests that the DKW in the core region of the 2F5
epitope EQELLELDKWASLWN (SEQ ID NO: 13) is essential for 2F5
binding. To test whether the potent neutralization of the TND_669S
envelope by 2F5 is also mediated though binding of the 2F5 mAb to
the core amino acids of the conventional 2F5 epitope, a
TND_669S/K665N mutant was made and its sensitivity to 2F5 and 4E10
mAbs was tested. Introduction of the K665N mutation into the
TND_669S envelope resulted in a fully 2F5-resistent phenotype while
the sensitivity of the envelope against 4E10 was not affected.
Ability of the 2F5 Peptides to Absorb the Neutralizing Activity of
the 2F5 mAb
To investigate the possible mechanisms involved in the ability of
the L669S substitution to increase the MPER neutralizing
sensitivity, peptides containing either the consensus 2F5 epitope
(2F5.sub.656-670) or the 2F5 epitope with the L669S substitution
(2F5.sub.656-670/L669S) were synthesized and subsequently tested
for their ability to absorb 2F5 mAb neutralizing activity. The 2F5
mAb was pre-absorbed with either the F5con or the F5mut peptide
prior to the neutralization assay. Surprisingly, F5mut did not
inhibit 2F5 mAb neutralization more potently than F5con. As show in
FIGS. 6A and 6B, both peptides inhibited 2F5 neutralization of the
TND_669S Env pseudovirus in a dose-dependent manner. However, F5con
is more efficient at inhibiting 2F5 neutralization, manifested by
comparable levels of inhibition achieved by 3 .mu.M of F5mut
(reduced the IC.sub.50 of the 2F5 mAb to 0.951 .mu.g/ml) and 0.3
.mu.M of F5con (reduced the IC.sub.50 of the 2F5 mAb to 0.911
.mu.g/ml) (FIG. 4B).
L669S Mutation Did not Increase the Binding Avidity of the 2F5 mAb
for its Epitope
To investigate the possibility that the L669S mutation enhances the
avidity of the 2F5 epitope to the 2F5 mAb, peptides containing
either the consensus 2F5 epitope (2F5.sub.656-670) or the 2F5
epitope with the L669S mutation (2F5.sub.656-670/L669S), along with
the scrambled version for each peptide, were tested in a BIAcore
SPR (surface plasmon resonance) assay for 2F5-binding
thermodynamics. The equilibrium dissociation constants (KD) for the
F5con and F5mut peptides were 11.0 and 28.1 nM, respectively (FIG.
5), indicating that F5con binds to 2F5 with a slightly higher
avidity than that of F5mut, although this 2.7-fold difference is
not significantly different. Binding ELISA data also confirmed that
there was no significant difference between the binding of the two
peptides by 2F5 mAb (FIG. 5). This suggests that other factors may
be involved in the differential sensitivity of the MPER sequences
such as a conformational change in the MPER that alters the
exposure of this region to neutralizing antibodies.
Binding of the Peptides to 2F5 mAb in Lipid Environment
In HIV-1 virus, MPER is in close proximity to the envelope lipid
bilayer. Direct binding SPR assay has shown that 2F5 mAb binds to
F5con and F5con peptides with comparable avidity. To further
examine the possible influence of the L669S substitution on binding
of the 2F5 mAb to its epitope in a lipid environment, a SPR binding
assay was performed using peptides anchored to
phospholipid-containing liposomes. As shown in FIG. 6, the peptide
containing the L669S substitution bound 2F5 mAb with a response
unit of 616.7 (background subtracted) at 10 seconds after the
injection was stopped, while the consensus 2F5 epitope bound 2F5
with a response unit of 494.6, indicating that in a lipid
environment, a 2F5 peptide with the L669S substitution does bind
stronger to 2F5 mAb than the consensus 2F5 mAb.
Fitness of TND_669S Virus is Greatly Impaired
To determine if the alteration in MPER structure resulted in a
fitness defect, the relative fitness of TND_669S and TND_669L
viruses was examined by a dual infection competition assay in
peripheral blood lymphocytes, using replication competent
recombinant viruses containing the NL4-3 backbone and the TND_669S
and TND_669L envelope sequences, respectively. With an input ratio
of 9:1 (TND_669S:TND_669L), the TND_669S virus was outgrown by the
TND_669L virus at 4 days post infection (FIG. 7), suggesting a
significant loss of fitness associated with the L669S mutation in
the TND 669S virus. The calculated relative fitness (1+S) is 1.86.
To further quantify the fitness differences, a ratio of 1:4
(TND_669S:TND_669L) was also examined and confirmed the lowered
fitness of the TND 669S virus (data not shown).
In summary, a mutation in the HIV-1 envelope, L669S, has been
identified that significantly increases the neutralization
sensitivity of the envelope to both 2F5 and 4E10 mAb
neutralization. The mean IC.sub.50 of the TND_669S and TND_669L
Env-pseudoviruses against mAbs 2F5 and 4E10 are 0.014 and 0.031
.mu.g/ml, respectively. In a study by Binley et al (J. Virol.
78:13232-13252 (2004)), where a panel of 93 HIV-1 strains were
examined for neutralizing sensitivity to various mAbs, most
isolates were neutralized by 2F5 and 4E10 with IC.sub.50 of 1-10
.mu.g/ml, while only 9 strains were neutralized at IC.sub.50<1
.mu.g/ml by 2F5 mAb, and 9 neutralized by 4E10 mAb at IC.sub.50 of
<1.0 .mu.g/ml. The IC.sub.50 of TND_669S against 2F5 and 4E10
mAbs was even lower than the most 2F5/4E10 mAb sensitive strain
(BUSxxxMNc), which was neutralized by 2F5 and 4E10 mAb with
IC.sub.50 values of 0.05 and 0.17 .mu.g/ml, respectively. In
comparison, the L669S mutation renders the envelope 4- and 5-fold
more sensitive to 2F5 and 4E10 mAb neutralization, respectively,
than the most sensitive virus previously reported.
A single amino acid mutation L669S is responsible for this specific
phenotype, as supported by site directed mutagenesis of the L669S
mutation into another primary isolate, QZ4734, which rendered the
QZ4734/L669S Env-pseudovirus more than 2 logs more sensitive to 2F5
mAb neutralization. To further confirm this, the serine at position
669 of the TND_669S was also mutated back to leucine resulting in
the loss of the ultra sensitivity observed in TND_669S
envelope.
Both TND_669S and TND 669L envelopes were obtained through bulk
PCR. Single genome amplification (SGA) was performed later but the
envelope sequences were not identified indicating that the L669S
mutation was not circulating in vivo. Additionally, the L669S
mutation results in a significant loss of fitness indicating that
even if present in natural infection, it would not have circulated
long because of its poor fitness level.
In an elegant alkaline-scanning mutagenesis study by Zwick et al,
J. Virol. 79:1252-1261 (2005), 13 out of 21 MPER Ala mutants were
more sensitive to 2F5 or 4E10 mAb, or both, than the parental MPER.
An L669A mutation in HIV-1 JR2 was 50- and 45-fold more sensitive
to neutralization by 2F5 and 4E10 mAbs, respectively, and was among
the most sensitivity-enhancing mutations. These findings, together
with present data, suggest that there may be some common mechanisms
shared by the 2F5 and 4E10 epitopes, such as the structure or the
accessibility of the MPER, that greatly affects Env sensitivity to
MPER neutralizing antibodies.
The mechanisms of the L669S substitution-associated increase in
HIV-1 envelope sensitivity to MPER neutralization warrants in depth
study because it sheds light on the neutralizing mechanisms of 2F5
and 4E10, and provides important information regarding immunogen
design to elicit these types of antibodies.
There are multiple ways through which this mutation may increase
neutralizing sensitivity. First, the mutation could have caused
dramatic changes in Env and affected the expression level of
functional Env spikes on viral particles. Neutralizing assays with
multiple other neutralizing agents showed that the increase in
neutralizing sensitivity of the TND_669S envelope is not a global
effect, making it unlikely that L669S mutation enhances
neutralizing sensitivity through changes in Env expression levels.
Secondly, this mutation could have changed the fusion kinetics of
gp41, resulting in a slower fusion process. Env with reduced fusion
kinetics have been shown to be more sensitive to 2F5 and 4E10
neutralization (Reeves et al, J. Virol. 79:4991-4999 (2005)). This
is unlikely since the sensitivity of the TND_669S envelope to T20
was only 3-fold that of the TND_669L envelope, suggesting the
fusion kinetics is not changed considerably by L669S mutation.
Thirdly, it is possible that the L669S mutation itself renders
higher avidity binding of the 2F5 mAb to the 2F5 epitope. This
hypothesis, however, is not supported by the surface plasmon
resonance (SPR) assay results for peptide binding to 2F5, where the
2F5 consensus peptide (containing the consensus 2F5 epitope
sequence) bound with slightly higher avidity than did the 2F5
mutatant peptide (containing the L669S mutation). Moreover, this
hypothesis can not explain the similar fold of increase in the
sensitivity of the TND_669S envelope to both 2F5 and 4E10 mAbs.
Fourthly, the L669S mutation could have caused dramatic
conformational change of Env, resulting in a more "open" MPER
structure, and thus allowing for easier access of antibodies
targeting 2F5 and 4E10. This hypothesis can very well explain the
similar magnitude of increase in sensitivity of the TND_669S
envelope to both 2F5 and 4E10 mAbs. The 447-52D sensitivity changes
associated with the L669S mutation (>161.times.) suggests that
the conformational change may have caused changes in the V3 loop as
well. Steric constraints for neutralizing antibodies targeting MPER
have been suspected by many groups. Several studies have observed
possible antagonism between 2F5 and 4E10 (Zwick et al, J. Virol.
79:1252-1261 (2005), Nelson et al, J. Virol. 81:4033-4043 (2007)),
suggesting that space limitation may be a factor affecting 2F5 and
4E10 neutralization of HIV virus. Interestingly, when 2F5 epitope
was inserted to MLV Env (Ou et al, J. Virol. 80:2539-2547 (2006)),
the Env with 2F5 epitope in surface unit is more than 10 times more
sensitive to 2F5 neutralization than the Env with 2F5 epitope in
the transmembrane unit. In addition, grafting 2F5 epitope into V1,
V2, V4 regions of HIV Env also was shown to increase the binding of
gp140 to 2F5 (Joyce et al, J. Biol. Chem. 277:45811-45820 (2002),
and grafting 2F5 and 4E10 epitopes to the MPER of HIV-2 has been
shown to be associated with substantial increase in
2F5-/4E10-neutralization sensitivity (Decker et al., presented at
the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone,
Colo., 2006), presumably through improved epitope accessibility.
These data reflected the influence of epitope accessibility on 2F5
sensitivity. The characteristic of TND_669S is in concordance with
a likely more "open" MPER structure.
The TND_669S isolate can be used to detect the presence of 2F5 and
4E10-like antibodies elicited by vaccination or natural infection
(studies to date have failed to detect 2F5 or 4E10 in HIV-1
infected patients and vaccines). An ultra-sensitive isolate can
provide crucial information as to whether or not 2F5/4E10 is
generated at extremely low levels during natural infection or
vaccination. Furthermore, the demonstration that a more exposed
MPER, as TND_669S envelope appears to have, has significant
applications for vaccine immunogen design.
EXAMPLE 2
Description of gp41MPER Peptide-Liposome Conjugates
FIG. 8 shows the amino acid sequences of each of the HIV-1 gp41MPER
peptides that can be conjugated to synthetic liposomes. While these
sequences have been used, longer gp41 sequences encompassing the
entirety of the Heptad Repeat 2 (HR2) region (aa 637-683), as well
as longer sequences involving the HR2 region as well as the HR1
region could be used (aa 549-602). The SP62 peptide presents the
2F5 mAb epitope while the MPER656 peptide includes both 2F5 and
4E10 mAb gp41 epitopes. (See WO 2008/127651.) Two variants of the
MPER peptide sequences include the SP62-L669S and the
MPER656-L669S. The L669S mutation was identified in an HIV-1
Envelope clone (TND_669S), obtained from a chronically infected
HIV-1+ subject, that was highly sensitive to neutralization by both
autologous and heterologous sera (see Example 1). TND_669S is
highly sensitive (with IC.sub.50 about 300-fold lower when compared
to TND_669L) to neutralization by both 2F5 and 4E10 mAbs (Shen J.
Virology 83: 3617-25 (2009)). The mutation resulted in more
favorable mAb binding kinetics with significantly slower off-rates
of the mAb 2F5-peptide liposome complex (SP62-L669S
peptide-liposomes). Tryptphan (W) immersion depth analysis of
SP62-liposomes suggested that the L669S substitution could alter
the orientation of the core 2F5 and 4E10 epitopes and make them
more accessible for B cell recognition. Thus, the use of L669S
substitution in both forms of liposomes with SP62-L669S and
MPER656-L669S peptides afford novel immunogens with favorably
exposed core MPER neutralizing epitopes and the potential for the
induction of neutralizing antibodies following immunization.
Description of gp41 MPER Peptide-Adjuvant Conjugates
Toll-like receptor ligands, shown in FIG. 9, were formulated in
liposomal forms with gp41MPER peptide immunogens. The ligands
referenced in FIG. 9 are examples only and other forms of TLR
agonists (Takeda et al, Annu. Rev. Immunol., 21:335-376 (2003)) can
be incorporated into similar liposomes as well.
The construction of Lipid A and R-848 containing MPER peptide
liposomes utilized the method of co-solubilization of MPER peptide
having a membrane anchoring amino acid sequence and synthetic
lipids 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE),
1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA) and Cholesterol at
mole fractions 0.216, 45.00, 25.00, 20.00 and 1.33 respectively
(Alam et al, J. Immunol. 178:4424-4435 (2007)). Appropriate amount
of MPER peptide dissolved in chloroform-methanol mixture (7:3 v/v),
Lipid A dissolved in Chloroform or R-848 dissolved in methanol,
appropriate amounts of chloroform stocks of phospholipids were
dried in a stream of nitrogen followed by over night vacuum drying.
Liposomes were made from the dried peptide-lipid film in phosphate
buffered saline (pH 7.4) using extrusion technology.
Construction of oligo-CpG complexed MPER peptide liposomes used the
cationic lipid 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-ethylphospho
choline (POEPC) instead of POPC. Conjugation of oCpG was done by
mixing of cationic liposomes containing the peptide immunogen with
appropriate amounts of oCpG stock solution (1 mg/ml) for the
desired dose.
Surface Plasmon Resonance (SPR) assay for the binding of 2F5 mAb to
its epitope in the peptide-liposome constructs revealed that
incorporation or conjugation of TLR adjuvants does not affect
binding of HIV neutralizing antibody 2F5. Strong binding of both
mAbs 2F5 and 4E10 were observed.
EXAMPLE 3
The long CDR H3 loops of MPER neutralizing mAbs 4E10 and 2F5 have a
hydrophobic face, postulated to interact with virion membrane
lipids (Ofek et al, J. Virol. 78:10724 (2004); Cardoso et al,
Immunity 22:163-173 (2005)). CDRH3 mutants of 4E10 (scFv) and 2F5
(IgG) have been constructed (see FIG. 13) and it has been found
that binding of neutralizing MPER mAbs occur sequentially and is
initiated by binding of mAbs to viral membrane lipids prior to
binding to prefusion intermediate state of gp41. 4E10 scFv bound
strongly to both nominal epitope peptide and a trimeric gp41 fusion
intermediate protein, but bound weakly to both HIV-1 and SIV
virions and thus indicating that 4E10 bound to viral membrane
lipids and not to the prefusion state of gp41. While alanine
substitutions at positions on the hydrophobic face of the CDR H3
loops of 4E10 (W100a/W100b/L100cA) showed similar binding to gp41
epitopes, the same substitutions disrutpted the ability of 4E10 to
bind to HIV-1 viral membrane (FIG. 14). 4E10 CDR H3 mutants that
bound to gp41 intermediate protein but did not bind to HIV-1 viral
membrane failed to neutralize HIV-1. Similarly, 2F5 CDR H3 mutants
with disruptions in binding to HIV-1 virions but not to gp41
epitope peptide, failed to neutralize HIV-1 (FIG. 14). Blocking of
HIV-1 neutralization activity of 4E10 by gp41 fusion intermediate
protein further suggested that 4E10 did not bind to viral prefusion
gp41. These results support the model that binding of neutralizing
MPER mAbs occurs sequentially and is initiated by binding of mAbs
to viral membrane lipids prior to binding to prefusion intermediate
state of gp41. An important implication of this result is that the
HIV-1 membrane constitutes an additional structural component for
binding and neutralization by 4E10 and 2F5. Thus, a lipid component
may be required for an immunogen to induce 4E10 and 2F5-like
antibody responses.
Thus, this strategy has the potential to modulate B cell tolerance,
target immunogens to responsive B cell subsets, and allow the
induction of polyreactive B cells that bind to phospholipids and
gp41MPER epitopes. When used in combination with TLR ligands, the
delivery of IFN-.alpha. in liposomes has the potential to allow
TLR-dependent activation of B cells from the autoreactive pool and
with the desired specificity for gp41MPER epitopes.
Description of Constructs:
The HIV-1 gp41MPER peptides (FIG. 8) can be conjugated to synthetic
liposomes as outlined above and described previously (Alam et al,
J. Immunol. 178:4424-4435 (2007)). Each of the sonicated MPER
peptide-liposomes can be prepared and then mixed with soluble
IFN.alpha. protein and then dried and rehydrated to encapsulate the
cytokine. After brief vortexing, the rehydrated liposomes with
encapsulated IFNa can be collected by ultracentrifugation for 30
min. In a first design, liposome is conjugated to either oCpG
(TLR9), MPL-A (TLR4) or R848 (TLR7/9) (FIG. 11). Each of these
adjuvanted liposome constructs can be prepared with each of the
listed MPER peptides shown in FIG. 8. A second design is shown in
FIG. 12 and includes multiple TLR ligands, TLR9+TLR4 and
TLR9+TLR7/8 incorporated into the same liposomes. The design of
these constructs can provide synergy in TLR triggering and
potentially enhance the potency of the TLR ligands in activating
polyreactive B cells.
The assessment of the presentation of MPER epitopes on the
adjuvanted liposome constructs can be done by SPR analysis of 2F5
and 4E10 mAb binding as described in FIG. 10.
EXAMPLE 4
Experimental Details
Representative data from two immunized animals show the application
of a prime/boost strategy for the induction of MPER specific
antibody responses following repeated immunizations with MPER
peptide liposomes (see FIG. 17). The animals were immunized at
alternating and at regular interval first with SP62 liposomes
(4.times.), and then with Env gp140 (2.times.) protein. The final
two immunizations include the full length MPER-656 liposomes (see
description of immunogens above). Binding responses in immunized
sera were measured by SPR analyses of binding to MPER peptide with
the shown sequence. Bleed samples from each immunized animals were
collected at the indicated post-bleed time points. Epitope mapping
of the immunized sera was done on the BIAcore A100 using
biotinylated alanine substituted MPER peptides with single amino
acid substitution of each MPER residue. Residues circled on top
indicate the critical residues (in red (underlined) with >50%
reduction in binding to alanine substituted peptide) required for
binding to the MPER peptide. Residues in blue (not underlined)
indicate residues with lower degree of involvement (<20-50%
reduction in binding).
Results
The presented experimental data shows the application of the
designed MPER liposomal immunogens in the induction of antibodies
that are targeted to the neutralizing epitopes on gp41 of HIV-1
Envelope protein. The data shows that the constructed MPER peptide
liposomes are immunogenic in small animals like guinea pigs and
non-human primates (NHP) and that the induced antibody responses
are specific for the core neutralizing epitope on gp41MPER. These
studies also demonstrate the application of prime-boost strategy in
enhancement of the MPER specific responses and in focusing of the
antibody responses to the core neutralizing epitopes that include
the 2F5 core residues DKW. In the presented immunization scheme,
the data shows a shift in the binding epitope in initial responses
from residues that are N-terminus to the core DKW to responses that
include all three residues of the core neutralizing epitope (DKW)
that are induced in later time points. Final immunizations with the
MPER liposomes resulted in focusing of the antibody responses to
the core DKW residues of the broad neutralizing mAb 2F5. These data
represents application of the design of MPER immunogens in
liposomal form for the induction of MPER specific antibodies in
experimental animals like guinea pigs (FIG. 17) and NHP (FIG. 18).
Such MPER immunogen designs can be candidates for human trials.
EXAMPLE 5
As shown in FIG. 18A, MPER specific binding responses were not
induced following priming with gp140 Env protein but were induced
following boosting with MPER liposomes. No binding responses to
MPER peptides were detected following multiple immunizations with
gp140 protein. Boosting of the same animals with MPER-656 liposomes
resulted in MPER specific responses that were specific for the 2F5
nominal epitope peptide.
As shown in FIG. 18B, epitope mapping of the antibody responses
show focusing of the response to the neutralizing 2F5 core residues
DKW. An initial broader specificity was focused to the DKW core
residues after the third immunization.
Binding data from four NHP immunized sera are shown. Binding
response measurements and epitope mapping experiments were done as
described in FIG. 17.
* * *
All documents and other information sources cited above are hereby
incorporated in their entirety by reference.
SEQUENCE LISTINGS
1
27125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1tagagccctg gaagcatcca ggaag 25224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ttgctacttg tgattgctcc atgt 24330DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3cacctaggca tctcctatgg
caggaagaag 30423DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 4gtctcgagat actgctccca ccc
23534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5ggataagtgg gcaagtttgt ggaattggtt tgac
34634DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6gtcaaaccaa ttccacaaac ttgcccactt atcc
34736DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7gaattattag aattggataa ctgggcaagt tcgtgg
36836DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ccacgaactt gcccagttat ccaattctaa taattc
36920PRTHuman immunodeficiency virus 1 9Gln Gln Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala1 5 10 15Ser Leu Trp Asn
201020PRTHuman immunodeficiency virus 1 10Gln Gln Glu Lys Asn Glu
Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala1 5 10 15Ser Ser Trp Asn
201137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Asp Lys Trp Ala1 5 10 15Ser Leu Trp Asn Tyr Lys Arg Trp Ile Ile
Leu Gly Leu Asn Lys Ile 20 25 30Val Arg Met Tyr Ser
351237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Asp Lys Trp Ala1 5 10 15Ser Ser Trp Asn Tyr Lys Arg Trp Ile Ile
Leu Gly Leu Asn Lys Ile 20 25 30Val Arg Met Tyr Ser 351315PRTHuman
immunodeficiency virus 1 13Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn1 5 10 15146PRTHuman immunodeficiency virus 1
14Glu Leu Asp Lys Trp Ala1 5156PRTHuman immunodeficiency virus 1
15Asn Trp Phe Asn Ile Thr1 51628PRTHuman immunodeficiency virus 1
16Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn1
5 10 15Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys 20
251728PRTHuman immunodeficiency virus 1 17Asn Glu Gln Glu Leu Leu
Glu Leu Asp Lys Trp Ala Ser Ser Trp Asn1 5 10 15Trp Phe Asn Ile Thr
Asn Trp Leu Trp Tyr Ile Lys 20 251852PRTHuman immunodeficiency
virus 1 18Asp Tyr Ile Tyr Ser Leu Leu Glu Asn Ala Gln Asn Gln Gln
Glu Arg1 5 10 15Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu Trp Asn 20 25 30Trp Phe Asp Ile Thr Lys Trp Leu Trp Tyr Ile Lys
Ile Phe Ile Met 35 40 45Ile Val Gly Gly 501952PRTHuman
immunodeficiency virus 1 19Asp Tyr Ile Tyr Ser Leu Leu Glu Asn Ala
Gln Asn Gln Gln Glu Lys1 5 10 15Asn Glu Gln Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu Trp Asn 20 25 30Trp Phe Asp Ile Ser Lys Trp Leu
Trp Tyr Ile Lys Ile Phe Ile Met 35 40 45Ile Val Gly Gly
502052PRTHuman immunodeficiency virus 1 20Gly Phe Ile Tyr Ser Leu
Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1 5 10 15Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 20 25 30Trp Phe Asp Ile
Ser Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 35 40 45Ile Val Gly
Gly 502152PRTHuman immunodeficiency virus 1 21Asp Phe Ile Tyr Ser
Leu Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1 5 10 15Asn Glu Gln Glu
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 20 25 30Trp Phe Asp
Ile Asn Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 35 40 45Ile Val
Gly Gly 502252PRTHuman immunodeficiency virus 1 22Asp Phe Ile Tyr
Ser Leu Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1 5 10 15Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Ser Trp Asn 20 25 30Trp Phe
Asp Ile Asn Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 35 40 45Ile
Val Gly Gly 502352PRTHuman immunodeficiency virus 1 23Asp Phe Ile
Tyr Ser Leu Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1 5 10 15Asn Glu
Gln Glu Leu Leu Glu Leu Asp Lys Arg Ala Ser Leu Trp Asn 20 25 30Trp
Phe Asp Ile Asn Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 35 40
45Ile Val Gly Gly 502452PRTHuman immunodeficiency virus 1 24Asp Tyr
Ile Tyr Ser Leu Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1 5 10 15Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 20 25
30Trp Phe Asp Ile Asn Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met
35 40 45Ile Val Gly Gly 502552PRTHuman immunodeficiency virus 1
25Asp Tyr Ile Tyr Ser Leu Leu Glu Asn Ala Gln Asn Gln Gln Glu Lys1
5 10 15Asn Glu Gln Glu Leu Leu Gly Leu Asp Lys Trp Ala Ser Leu Trp
Asn 20 25 30Trp Ser Asp Ile Asn Lys Trp Leu Trp Tyr Arg Lys Ile Phe
Ile Met 35 40 45Ile Val Gly Gly 502631PRTHuman immunodeficiency
virus 1 26Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala1 5 10 15Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn Trp Leu Tyr
Ile Lys 20 25 302722DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 27tcgtcgttgt cgttttgtcg tt
22
* * * * *